人源血管紧张素转化酶-C结构域在毕赤酵母中的表达

血管紧张素转化酶 (ACE，EC3.4.15.1) 在调节血压方面具有重要作用，研究证实，ACE-C结构域是体内使血管紧张素I (AngI) 分解的主要活性位点。PCR扩增ACE-C结构域基因片段，克隆至分泌表达载体pPIC9K，转化毕赤酵母GS115，阳性克隆再次电转，用G418筛选高拷贝的酵母菌落进行表达条件优化，获得0.5 g/L的蛋白表达量及7.178 U/mL的酶活力。经Ni+亲和层析纯化，获得纯度大于97％的目的蛋白。Captopril对酶的抑制试验证明ACE-C结构域可望成为新一代抗高血压药物ACE抑制剂筛选的理想酶靶。

Expression of human angiotensin converting enzyme-C domain in Pichia pastoris

Angiotensin I-converting enzyme (ACE, EC3.4.15.1) plays an important role in regulating blood pressure. The C-domain of ACE has been identified as the main catalytic site of angiotensin I cleavage in vivo. The ACE gene fragment of the C-domain was amplified by PCR and cloned into the pPIC9K secretory expression plasmid. The recombinant plasmid was transformed into Pichia pastoris strain GS115. Positive clones were selected and subject to electroporation. Antibiotic G418 was used for the screening of multicopy inserts. After optimization of the expression system, the protein yield reached 0.5 g/L by flask-shaking culture fermentation, and enzyme activity reached 7.178 U/mL in the fermentation supernatant. The purity of the target protein obtained was 97% after Ni+ affinity chromatography. Enzyme inhibitory activity assay using Captopril showed that it is promising to use ACE-C domain as new generation of target for screening ACE inhibitor antihypertensive drugs.